CRFR1 selective ligands

ABSTRACT

CRF peptide analogs that bind to CRFR1 with an affinity far greater than they bind to CRFR2. These analogs exhibit CRF agonist activity. One exemplary analog that may be made by solid-phase synthesis is (cyclo 31-34)[Ac-Pro 4 , D-Phe 12 , Nle 21,38 , Glu 31 , Lys 34 ]-r/hCRF(4-41).

This application is a continuation of PCT US02/24238, filed Jul. 30,2002, which application claimed priority from U.S. ProvisionalApplication Ser. No. 60/309,504, filed Aug. 1, 2001, the disclosures ofwhich are expressly incorporated hereinafter by reference.

This invention was made with Government support under grant numberP01-DK-26741 awarded by the National Institutes of Health. The U.S.Government has certain rights in this invention.

This invention is generally directed to peptides and to thepharmaceutical treatment of mammals using such peptides. Morespecifically, the invention relates to peptide analogs to thehentetracontapeptide CRF which are selective to one family of CRFreceptors, to pharmaceutical compositions containing such CRF analogs,to methods of treatment of mammals using such CRF analogs, and tomethods of screening for new drugs using such peptides.

BACKGROUND OF THE INVENTION

Ovine CRF (oCRF) was characterized in 1981 as a 41-residue amidatedpeptide. oCRF lowers blood pressure in mammals when injectedperipherally and stimulates the secretion of ACTH and β-endorphin. RatCRF (rCRF) was later isolated, purified and characterized; it was foundto be a homologous, amidated hentetracontapeptide as described in U.S.Pat. No. 4,489,163. The amino acid sequence of human CRF (hCRF) wasdetermined to be the same as that of rCRF. When given intravenously(iv),hCRF and oCRF have been reported to cause vasodilation of the mesentericarteries so as to lower blood pressure in mammals and also instimulating the secretion of ACTH and β-endorphin. However, whenadministered intracerebroventricularly(icv), there is an elevation ofheart rate and mean arterial blood pressure, which are secondary toactivation of the sympathetic nervous system.

Although originally isolated and characterized on the basis of its rolein this hypothalamopituitary-adrenal (HPA) axis, CRF has been found tobe distributed broadly throughout the central nervous system as well asin extraneural tissues, such as the adrenal glands, placenta and testes,where it may also act as a paracrine regulator or a neurotransmitter.Moreover, the likely involvement of CRF in affective disorders, such asanxiety, depression, alcoholism and anorexia nervosa, and in modulatingreproduction and immune responses suggests that changes in CRFexpression may have important physiological and pathophysiologicalconsequences. For example, perturbations in the regulatory loopscomprising the HPA axis often produce chronically elevated levels ofcirculating glucocorticoids; such patients display the physicalhallmarks of Cushing's syndrome, including truncal obesity,muscle-wasting, and reduced fertility.

In addition to its role in mediating activation of thehypothalamic-pituitary-adrenal, CRF has also been shown to modulateautonomic and behavioral changes, some of which occur during the stressresponse. Many of these behavioral changes have been shown to occurindependently of HPA activation in that they are not duplicated bydexamethasone treatment and are insensitive to hypophysectomy. Inaddition, direct infusion of CRF into the CNS mimics autonomic andbehavioral responses to a variety of stressors. Because peripheraladministration of CRF fails to affect certain of these changes, itappears that CRF exhibits a direct brain action with respect to suchfunctions, which include appetite suppression, increased arousal andlearning ability.

As a result of the extensive anatomical distribution and multiplebiological actions of CRF, this regulatory peptide is believed to beinvolved in the regulation of numerous biological processes. CRF hasalso been implicated in the regulation of inflammatory responses.Although it has been observed that CRF plays a pro-inflammatory role incertain animal models, CRF appears to suppress inflammation in others byreducing injury-induced increases in vascular permeability.

Recent clinical data have implicated corticotropin-releasing factor(“CRF”) in neuropsychiatric disorders and in neurodegenerative diseases,such as Alzeimer's disease. Alzheimer's disease is a neurodegenerativebrain disorder which leads to progressive memory loss and dementia. Bycurrent estimates, over two million individuals in the United Statessuffer from this disease. In particular, several lines of evidence haveimplicated CRF in Alzheimer's disease (AD) (Behan et al., Nature378(16):284, 1995). First, there are dramatic (greater than 50%)decreases in CRF (Bissette et al., JAMA 254:3067, 1985; DeSouza et al.,Brain Research 397:401, 1986; Whitehouse et al., Neurology 37:905, 1987;DeSouza, Hospital Practice 23:59, 1988; Nemeroff et al., Regul. Peptides25:123, 1989) and reciprocal increases in CRF receptors (DeSouza et al.,1986; DeSouza, 1988) in cerebrocortical areas that are affected in AD,while neither CRF nor CRF receptors are quantitatively changed innon-affected areas of the cortex (DeSouza et al., 1986). Second,chemical affinity crosslinking studies indicate that the increased CRFreceptor population in cerebral cortex in AD have normal biochemicalproperties (Grigoriadis et al., Neuropharmacology 28:761, 1989).Additionally, observations of decreased concentrations of CRF in thecerebrospinal fluid (Mouradian et al., Neural Peptides 8:393, 1986; Mayet al., Neurology 37:535, 1987) are significantly correlated with theglobal neuropsychological impairment ratings, suggesting that greatercognitive impairment is associated with lower CRF concentrations incerebrospinal fluid (Pomara et al., Biological Psychiatry 6:500, 1989).

Available therapies for the treatment of dementia are severely limited.Tacrine.™., a recently approved drug, leads to only marginal memoryimprovement in Alzheimer's patients, and has the undesirable side effectof elevating liver enzymes. Alterations in brain CRF content have alsobeen found in Parkinson's disease and progressive supranuclear palsy,neurological disorders that share certain clinical and pathologicalfeatures with AD. In cases of Parkinson's disease, CRF content isdecreased and shows a staining pattern similar to cases of AD(Whitehouse et al., 1987; DeSouza, 1988). In progressive supranuclearpalsy, CRF is decreased to approximately 50% of control values infrontal, temporal, and occipital lobes (Whitehouse et al., 1987;DeSouza, 1988).

Some depressive disorders are also associated with decreased levels ofCRF. Patients in the depressive state of seasonal depression and in theperiod of fatigue in chronic fatigue syndrome demonstrate lower levelsof CRF in the cerebrospinal fluid (Vanderpool et al., J Clin.Endocrinol. Metab. 73:1224, 1991). Although some depressions have a highimprovement rate and many are eventually self-limiting, there are majordifferences in the rate at which patients recover. A major goal oftherapy is to decrease the intensity of symptoms and hasten the rate ofrecovery for this type of depression, as well as preventing relapse andrecurrence. Anti-depressants are typically administered, but severe sideeffects may result (e.g., suicidality with fluoxetine, convulsions withbupropion). (See Klerman et al. in Clinical Evaluation of PsychotropicDrugs: Principles and Guidelines, R. F. Prien and D. S. Robinson (eds.),Raven Press, Ltd. N.Y., 1994, p. 281.)

Hypoactivation of the stress system as manifested by low CRF levels mayplay a role in other disorders as well. For examples, some forms ofobesity are characterized by a hypoactive hypothalamic-pituitary-adrenalaxis (Kopelman et al., Clin. Endocrinol (Oxford) 28:15, 1988; Bernini etal., Horm. Res. 31:133, 1989), some patients with post-traumatic stresssyndrome have low cortisol excretion (Mason et al., J. Neu. Men. Dis.174:145, 1986), and patients undergoing withdrawal from smoking havedecreased excretion of adrenaline and noradrenaline, as well asdecreased amounts of cortisol in blood (West et al., Psychopharmacology84:141, 1984; Puddy et al., Clin. Exp. Pharmacol. Physiol. 11:423,1984). These manifestations all point to a central role for CRF in thesedisorders because CRF is the major regulator of thehypothalamic-pituitary-adrenal axis. Treatments for these disorders havepoor efficacy. For example, the most effective approach to treatment ofobesity is a behavior-change program. However, few participants reachgoal weight and the relapse rate is high (see Halmi et al. in ClinicalEvaluation of Psychotropic Drugs: Principles and Guidelines, R. F. Prienand D. S. Robinson (eds.), Raven Press, Ltd. New York, 1994, p. 547).

In view of the deficiencies in treatments for such disorders anddiseases, more effective treatments are needed. The present inventionexploits the correlation of reduced levels of CRF with variousneuro-physiologically based disorders and diseases to effectively treatsuch diseases by increasing levels of free CRF, and further providesother related advantages. Because these actions are mediated by CRFR2,CRFR2-selective analogs are preferred over non-selective analogs due tothe possible side effects resulting from activation of other CRFreceptors.

CRF agonists containing D-isomers of α-amino acids were developed, suchas those shown in U.S. Pat. No. 5,109,111. Other agonists of CRF aredisclosed in U.S. Pat. No. 5,278,146. Cyclic CRF agonists exhibitingbiopotency were later developed as disclosed in U.S. Pat. Nos. 5,824,771and 5,844,074.

CRF-R is used to refer to a family of receptor protein subtypes whichparticipate in the G-protein-coupled response of cells to CRF. CRF-Rsare coupled by heterotrimeric G-proteins to various intracellularenzymes, ion channels, and transporters. The G-proteins associate withthe receptor proteins at the intracellular face of the plasma membrane.An agonist binding to a CRF-R catalyzes the exchanges of GTP for GDP onthe α-subunit (G-protein “activation”), resulting in its dissociationand stimulation of one (or more) of the various signal-transducingenzymes and channels. G-protein preferentially stimulates particulareffectors, and the specificity of signal transduction may be determined,therefore, by the specificity of G-protein/receptor interaction. CRF-Rproteins mediate signal transduction through the modulation of adenylatecyclase and perhaps through PI turnover. For example, when CRF binds toand activates the CRF-R, adenylate cyclase causes an elevation in thelevel of intracellular cAMP. An effective bioassay for evaluatingwhether a test compound is capable of elevating intracellular cAMP iscarried out by culturing cells containing cDNA which expresses CRFreceptor proteins in the presence of a potential agonist or antagonistwhose ability to modulate signal transduction activity of CRF receptorprotein is sought to be determined. Such transformed cells are monitoredfor either an increase or decrease in the level of intracellular cAMPwhich provides a determination of the effectiveness of the potentialagonist or antagonist. Methods for measuring intracellular levels ofcAMP, or measuring cyclase activity, are well known in the art.

The physiological actions of CRF are mediated through activation of atleast two high affinity receptors, CRFR1 and CRFR2, which are members ofthe seven-transmembrane family of receptors [Chen R., et al, P.N.A.S.,90:8967-8971 (1993), Perrin, M., et al., P.N.A.S, 92:2969-2973 (1995),Lovenberg, T., et al., P.N.A.S., 92:836-840 (1995), K. D. Dieterich etal. Exp. Clin. Endocrinol. Diabetes (1997) 105:65-82 and J. Spiess etal. Trends Endocrinol. Metab. (1998) 9:140-145]. Evidence fromtransgenic knockouts [A. Contarino et al., Brain Res. (1999) 835:1-9, G.W. Smith et al., Neuron (1998) 20:1093-1102 and P. Timpl et al., NatureGenet. (1998) 19:162-166], antisense oligonucleotide studies [S. C.Heinrichs et al., Regul. Pept. (1997) 71:15-21, G. Liebsch et al., J.Psychiatric Res. (1999) 33:153-163 and T. Skutella et al., Neuroscience(1998) 85: 795-805] and CRFR1 antagonists [K. E. Habib et al., Proc.Natl. Acad. Sci. USA (2000) 97:6079-6084., J. Lundkvist et al., Eur. J.Pharmacol. (1996) 309:195-200., R. S. Mansbach et al., Eur. J.Pharmacol. (1997) 323:21-26 and S. C. Weninger et al., Proc. Natl. Acad.Sci. USA (1999) 96:8283-8288] provide evidence for the involvement ofCRFR1 in mediating the anxiogenic effects of CRF.

The CRF2 was identified more recently [T. Kishimoto et al., Proc. Natl.Acad. Sci. USA (1995) 92:1108-1112, W. A. Kostich et al., Mol.Endocrinol. (1998) 12:1077-1085, T. W. Lovenberg et al., Proc. Natl.Acad. Sci. USA (1995) 92:836-840. and M. Perrin et al., Proc. Natl.Acad. Sci. USA (1995) 92:2969-2973] and exists as at least three splicevariants. CRFR1 and CRFR2 subtypes are 70% homologous in their aminoacid sequences but appear to be pharmacologically [D. P. Behan et al.,Mol. Psychiatry (1996) 1:265-277. and K. D. Dieterich et al., Exp. Clin.Endocrinol. Diabetes (1997) 105:65-82.] and anatomically distinct [D. T.Chalmers et al., J. Neurosci. (1995) 15:6340-6350. and D. H. Rominger etal., J. Pharmacol. Exp. Ther. (1998) 286:459-468].

CRFR1 is distributed throughout the brain and the sensory and motorrelay sites, whereas CRFR2 is expressed in regions of the body wherethere is little or no expression of CRFR1, such as peripheral sites,e.g. the blood vessels, the heart, the GI tract, the lungs and the skin.In addition, while CRFR1 expression is very high in neocortical,cerebellar, and sensory relay structures, CRFR2 expression is generallyconfined to subcortical structures. Within the pituitary gland, CRFR2mRNA is detectable at low levels in scattered cells while CRF1 receptormRNA is readily detectable in anterior and intermediate lobes.

This heterogeneous distribution of CRFR1 and CRFR2 mRNA suggestsdistinctive functional roles for each receptor in CRF-related systems.CRFR1 may be regarded as the primary neuroendocrine pituitary CRFreceptor and important in cortical, cerebellar and sensory roles of CRF.

Both CRFR1 and CRFR2 were found in the pituitary and throughout theneocortex (especially, in prefrontal, cingulate, striate, and insularcortices), amygdala, and hippocampal formation of primates. In primates,both CRFR1 and CRFR2 may be involved in mediating the effects of CRF oncognition, behavior, and pituitary-adrenal function. The presence ofCRFR1 (but not CRFR2) within the locus coeruleus, cerebellar cortex,nucleus of the solitary tract, thalamus, and striatum and of CRFR2 (butnot CRFR1) in the choroid plexus, certain hypothalamic nuclei, thenucleus prepositus, and the nucleus of the stria terminalis suggeststhat each receptor subtype also may have distinct functional roleswithin the primate central nervous system. See, e.g., Sanchez et al., J.Comp. Neurol. 408:365-377.

CRF has been widely implicated as playing a major role in modulating theendocrine, autonomic, behavioral and immune responses to stress. Therecent cloning of multiple receptors for CRF as well as the discovery ofnon-peptide receptor antagonists for CRF receptors have begun a new eraof CRF study. Presently, there are five distinct targets for CRF withunique cDNA sequences, pharmacology and localization. These fall intothree distinct classes, encoded by three different genes and have beentermed the CRFR1 and CRFR2 (belonging to the superfamily of G-proteincoupled receptors) and CRF-binding protein.

Expression of these receptors in mammalian cell lines has made possiblethe identification of non-peptide, high affinity, selective receptorantagonists. While the natural mammalian ligands oCRF and r/hCRF havehigh affinity for the CRFR1 subtype, they have lower affinity for theCRFR2 family making them ineffective labels for CRF2. [¹²⁵I]Sauvaginehas been characterized as a high affinity ligand for both the CRFR1 andthe CRFR2 subtypes and has been used in both radioligand binding andreceptor autoradiographic studies as a tool to aid in the discovery ofselective small molecule receptor antagonists. A number of non-peptideCRFR1 antagonists that can specifically and selectively block the CRFR1subtype have recently been identified. Compounds such as CP 154,526, NBI27914 and Antalarmin inhibit CRF-stimulation of cAMP or CRF-stimulatedACTH release from cultured rat anterior pituitary cells. Furthermore,when administered peripherally, these compounds compete for ex vivo[¹²⁵I]sauvagine binding to CRFR1 in brain sections demonstrating theirability to cross the blood-brain-barrier. In in vivo studies, peripheraladministration of these compounds attenuate stress-induced elevations inplasma ACTH levels in rats demonstrating that CRFR1 can be blocked inthe periphery. Furthermore, peripherally administered CRFR1 antagonistshave also been demonstrated to inhibit CRF-induced seizure activity.These data clearly demonstrate that non-peptide CRFR1 antagonists, whenadministered systemically, can specifically block central CRFR1 andprovide tools that can be used to determine the role of CRFR1 in variousneuropsychiatric and neurodegenerative disorders. In addition, thesemolecules will prove useful in the discovery and development ofpotential orally active therapeutics for these disorders. McCarthy etal., Curr Pharm Des. (1999) 5(5):289-315.

Because the CRFR1 control different functions than the CRFR2, it wouldbe valuable to be able to regulate one family of receptors withoutsignificantly affecting the other family. oCRF and rCRF bindsubstantially similarly to both CRFR1 and CRFR2 families. A. Ruhmann etal. P.N.A.S., 95, 15264-15269 (Dec. 1998) reported that [D-Phe¹¹,His¹²]-sauvagine(11-40) was an antagonist that acted selectively withrespect to CRFR2 and exhibited competitive antagonism equal to about 30%of that of the then best antagonist for CRFR1 and close to equalantagonism for CRFR2 compared to this previously best reported compound.Thereafter, the search has continued for CRF analogs that will bebioactive as CRFR1-selective agonists and also for analogs that willserve as effective competitive antagonists to modulate the activation ofCRFR1 while having less effect upon CRFR2.

SUMMARY OF THE INVENTION

A class of CRF peptides which are ligands of CRFR1 has now been foundwhich are analogs of hCRF/oCRF and preferably have a cyclizing bondbetween the residues that correspond to residues 31 and 34 of the nativeCRF molecule, which cyclizing bond is preferably an amide linkagebetween side chains of the amino acid residues in those positions. TheC-terminus of the molecules is the native amide; however the N-terminusis preferably shortened by elimination of the first 3 residues and byacylation of the residue in position 4 at the N-terminus. The comparablelinear peptides also show selectivity and high binding strength to theCRFR1; however, they are not believed to be as biopotent. Usingtechniques well known in this art, selective agonists can be transformedinto CRF antagonists that will selectively block the CRFR1 by retainingthe disclosed cyclic portion of the core structure.

Pharmaceutical compositions in accordance with the invention includesuch CRFR1 ligands or nontoxic addition salts thereof that are dispersedin a pharmaceutically acceptable liquid or solid carrier. Suchformulation is facilitated because of their high solubility atphysiological pH. The administration of such peptides orpharmaceutically acceptable addition salts thereof to mammals,particularly humans, in accordance with the invention may be carried outfor the regulation of secretion of ACTH, β-endorphin, β-lipotropin,corticosterone and other products of the pro-opiomelanocortin (POMC)gene and/or for affecting mood, behavioral and gastrointestinalfunctions and autonomic nervous system activities. For example, theseCRF analogs may be administered to increase ACTH levels to treat shockand like conditions. Very generally, administration of a compound of thepresent invention can be used to treat a wide variety of disorders orillnesses, particularly associated with CRFR1. In particular, thecompounds of the present invention may be administered to an animal forthe treatment of depression, anxiety disorder, panic disorder,obsessive-compulsive disorder, reactive hypertension, anorexia nervosa,bulimia, irritable bowel syndrome, stress-induced immune suppression andepilepsy.

The peptides also provide the basis for valuable methods for drugscreening for even more potent molecules which bind to and/or activateCRF receptors because of their high affinity for CRF receptors, andradioactive analogs can be used as tracers that bind selectively toCRFR1 and are valuable for high throughput screening purposes.

In one particular aspect, the invention provides a 38-residue CRFR1ligand peptide which binds to CRFR1 with an affinity substantiallygreater than it binds to CRFR2, which peptide has the following formula,or a nontoxic salt thereof:

Y₁-Pro-Pro-R₆-Ser-R₈-Asp-R₁₀-R₁₁-D-Phe-R₁₃-R₁₄-R₁₅-Arg-R₁₇-R₁₈-R₁₉-R₂₀-R₂₁-R₂₃-R₂₄-R₂₅-R₂₆-R₂₇-R₂₈-R₂₉-Gln-Glu-R₃₂-R₃₃-R₃₄-Arg-R₃₆-R₃₇-R₃₈-R₃₉-R₄₀-R₄₁-NH₂wherein Y₁ is an acyl group having not more than 15 carbon atoms or isradioiodinated tyrosine; R₆ is Ile, Met or Nle; P₈ is Leu or Ile; R₁₀ isLeu or CML; R₁₁ is Thr or Ser; R₁₃ is His, Tyr or Glu; R₁₄ is CML orLeu; R₁₅ is CML or Leu; R₁₇ is Glu, CML, Asn or Lys; R₁₈ is Val, CML,Nle or Met; R₁₉ is CML, Leu or Ile; R₂₀ is Glu, D-Glu or His; R₂₁ isNle, Leu, CML or Met; R₂₂ is Ala, D-Ala, Aib, Thr, Asp or Glu; R₂₃ isArg or Lys; R₂₄ is Ala, Gln, Ile, Asn, CML or Aib; R₂₅ is Asp or Glu;R₂₆ is Gln, Asn or Lys; R₂₇ is CML, Glu, Gln or Leu; R₂₈ is Ala, Lys,Arg or Aib; R₂₉ is Gln, Aib or Glu; R₃₂ is Aib or an L- or D-isomer of anatural α-amino acid other than Cys; R₃₃ is Aib or an L- or D-isomer ofSer, Asn, Leu, Ala, CML or Ile; R₃₄ is Lys or Orn; R₃₆ is Lys, Orn, Arg,Har, CML or Leu; R₃₇ is CML, Leu, Nle or Tyr; R₃₈ is Nle, Met, CML orLeu; R₃₉ is Glu, Aib or Asp; R₄₀ is Ile, Aib, CML, Thr, Glu, Ala, Val,Leu, Nle, Phe, Nva, Gly or Gln; and R₄₁ is Ala, Aib, Ile, CML, Gly, Val,Leu, Nle, Phe, Nva or Gln; provided that a cyclizing bond may existbetween Glu in position 31 and R₃₄ and provided further that D-2Nal orD-Leu may be substituted for D-Phe.

In another particular aspect, the invention provides a 38-residue CRFR1ligand peptide which binds to CRFR1 with an affinity substantiallygreater than it binds to CRFR₂, which peptide has the formulaY₁-Pro-Pro-A-D-Xaa-B-Glu-Xa_(a)-Xaa_(b)-Xaa_(c)-C-NH₂ wherein Y₁ is anacyl group having not more than 15 carbon atoms or is radioiodinatedtyrosine; A is a sequence of 6 amino acid residues that is found betweenPro in the 5-position and Phe in the 12-position of r/hCRF or thecorresponding sequence of another peptide of the CRF family; D-Xaa isD-Phe, D-2Nal or D-Leu; B is a sequence of 18 amino acid residues thatis found between Phe in the 12-position and Ala in position-31 of r/hCRFor the corresponding sequence of another peptide of the CRF family;Xaa_(a) is any L- or D-natural α-amino acid other than Cys or is Aib;Xaa_(b) is Aib or an L- or D-isomer of Ser, Asn, Leu, Ala, CML or Ile;Xaa_(c) is either Lys or Orn, the side chain of which may be linked inan amide cyclizing bond to that of Glu; and C is a sequence of the last7 amino acid residues of the C-terminal portion of any peptide of theCRF family; provided that Nle or Leu may be substituted for Met in A, Band/or in C.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Because the CRF receptor ligands of the present invention demonstrateactivity at the CRFR1 receptor site, they may be used as therapeuticagents for the treatment of a wide range of disorders or illnessesincluding endocrine, psychiatric, and neurologic disorders or illnesses.More specifically, the CRF receptor ligands of the present invention maybe useful in treating physiological conditions or disorders arising fromthe hypersecretion of CRF. Because CRF is believed to be a pivotalneurotransmitter that activates and coordinates the endocrine,behavioral and automatic responses to stress, the CRF receptor ligandsof the present invention can be used to treat neuropsychiatricdisorders. Neuropsychiatric disorders which may be treatable by the CRFreceptor ligands of this invention include affective disorders such asdepression; anxiety-related disorders such as generalized anxietydisorder, panic disorder, obsessive-compulsive disorder, abnormalaggression, cardiovascular abnormalities such as unstable angina andreactive hypertension; and feeding disorders such as anorexia nervosa,bulimia, and irritable bowel syndrome. CRF receptor ligands may also beuseful in treating stress-induced immune suppression associated withvarious diseases states, as well as stroke. Other uses of the CRFreceptor ligands of this invention include treatment of inflammatoryconditions (such as rheumatoid arthritis, uveitis, asthma, inflammatorybowel disease and G.I. motility), Cushing's disease, infantile spasms,epilepsy and other seizures in both infants and adults, and varioussubstance abuse and withdrawal (including alcoholism).

In another embodiment of the invention, pharmaceutical compositionscontaining one or more CRF receptor ligands are disclosed. For thepurposes of administration, the compounds of the present invention maybe formulated as pharmaceutical compositions. Pharmaceuticalcompositions comprise a CRF receptor ligand of the present invention anda pharmaceutically acceptable carrier and/or diluent. The CRF receptorligand should be present in the composition in an amount which iseffective to treat a particular disorder, that is, in an amountsufficient to achieve desired CRF activity, and preferably withacceptable toxicity to the patient. Preferably, the pharmaceuticalcompositions of the present invention may include a CRF receptor ligandin an amount from 0.1 mg to 250 mg per dosage depending upon the routeof administration, and more preferably from 1 mg to 60 mg. Appropriateconcentrations and dosages can be readily determined by one skilled inthe art.

The nomenclature used to define the peptides is that specified bySchroder & Lubke, “The Peptides”, Academic Press (1965) wherein, inaccordance with conventional representation, the amino group appears tothe left and the carboxyl group to the right. The standard 3-letterabbreviations are used to identify the alpha-amino acid residues, andwhere the amino acid residue has isomeric forms, it is the L-form of theamino acid that is represented unless otherwise expressly indicated,e.g. Ser=L-serine, Orn=L-ornithine, Nle=L-norleucine, Nva=L-norvaline,Agl=aminoglycine, Abu=L-2-aminobutyric acid, Dbu=L-2,4-diaminobutyricacid, Dpr=L-2,3-diaminopropionic acid, Hly=L-homolysine andHar=L-homoarginine. In addition the following abbreviations are used:CML=C^(α)CH₃-L-leucine; Aib=C^(α)CH₃-L-alanine or 2-aminoisobutyricacid; Nal=L-α-(1- or 2-naphthyl) alanine, Pal=L-β-(2-,3- or4-pyridyl)alanine, Cpa=L-(2-, 3-, or 4-chloro) phenylalanine,Aph=L-(2-,3- or 4-amino) phenylalanine, Amp=(2-, 3- or 4-aminomethyl)phenylalanine, and Nic=3-carboxypyridine (or nicotinic acid).

Generally, the CRFR1 ligands include a D-isomer in the 12-position,preferably include a cyclizing linkage between the residues in the31-position and the 34-position, and have the following amino acidsequence, or are equivalent nontoxic salts thereof:

Y₁-Pro-Pro-R₆-Ser-P₈-Asp-R₁₀-R₁₁-D-Phe-R₁₃-R₁₄-R₁₅-Arg-R₁₇-R₁₈-R₁₉-R₂₀-R₂₁-R₂₂-R₂₃-R₂₄-R₂₅-R₂₆-R₂₇-R₂₈-R₂₉-Gln-Glu-R₃₂-R₃₃-R₃₄-Arg-R₃₆-R₃₇-R₃₈-R₃₉-R₄₀R₄₁-NH₂wherein Y₁ is an acyl group having not more than 15 carbon atoms or isradioiodinated tyrosine; R₆ is Ile, Met or Nle; R₈ is Leu or lie; R₁₀ isLeu or CML; R₁₁ is Thr or Ser; R₁₃ is His, Tyr or Glu; R₁₄ is CML orLeu; R₁₅ is CML or Leu; R₁₇ is Glu, CML, Asn or Lys; R₁₈ is Val, CML,Nle or Met; R₁₉ is CML, Leu or Ile; R₂₀ is Glu, D-Glu or His; R₂₁ isNle, Leu, CML or Met; R₂₂ is Ala, D-Ala, Aib, Thr, Asp or Glu; R₂₃ isArg or Lys; R₂₄ is Ala, Gln, Ile, Asn, CML or Aib; R₂₅ is Asp or Glu;R₂₆ is Gln, Asn or Lys; R₂₇ is CML, Glu, Gln or Leu; R₂₈ is Ala, Lys,Arg or Aib; R₂₉ is Gln, Aib or Glu; R₃₂ is Aib or an L- or D-isomer of anatural α-amino acid other than Cys; R₃₃ is Aib or an L- or D-isomer ofSer, Asn, Leu, Ala, CML or Ile; R₃₄ is Lys or Orn; R₃₆ is Lys, Orn, Arg,Har, CML or Leu; R₃₇ is CML, Leu, Nle or Tyr; R₃₈ is Nle, Met, CML orLeu; R₃₉ is Glu, Aib or Asp; R₄₀ is Ile, Aib, CML, Thr, Glu, Ala, Val,Leu, Nle, Phe, Nva, Gly or Gln; and R₄, is Ala, Aib, Ile, CML, Gly, Val,Leu, Nle, Phe, Nva or Gln; provided that a cyclizing bond may existbetween Glu in position 31 and R₃₄ and provided further that D-2Nal orD-Leu may be substituted for D-Phe.

A particularly preferred group of CRF agonists has the amino acidsequence (including nontoxic salts thereof):

(cyclo31-34)Y₁-Pro-Pro-R₆-Ser-R₈-Asp-Leu-R₁₁-D-Phe-His-R₁₄-Leu-Arg-Glu-R₁₈-Leu-R₂₀-Nle-R₂₂-R₂₃-Ala-R₂₅-Gln-Leu-Ala-R₂₉-Gln-Glu-R₃₂-R₃₃-R₃₄-Arg-R₃₆-R₃₇-Nle-R₃₉-R₄₀-R₄₁-NH₂wherein Y₁ is an acyl group having not more than 7 carbon atoms; R₂₀ isGlu or D-Glu; R₂₂ is Ala or Thr; R₂₉ is Gln or Glu; R₃₂ is His, Aib,Ala, Gly, Leu, Gln or Glu; R₃₆ is Lys or Leu; R₃₇ is Leu or CML; R₃₉ isGlu or Asp; R₄₀ is Ile, CML or Glu; and R₄₁ is Ile, Aib or Ala; with theremaining variables being defined as above.

Specific analogs which are considered to be particularly biopotent fromthe standpoint of increasing ACTH levels are:

-   cyclo(31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), Glu³¹,    Lys³⁴]-r/hCRF(4-41);-   (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), Glu³¹,    Lys³⁴]-oCRF(4-41);-   (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), Glu³¹, Aib³³,    Lys³⁴]-r/hCRF(4-41);-   (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), CML²⁷, Glu³¹,    Lys³⁴]-r/hCRF(4-41);-   (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), CML^(27,40), Glu³¹,    Lys³⁴]-r/hCRF(4-41); and-   (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), CML^(27,40), Glu³¹,    Aib³³, Lys³⁴]-r/hCRF(4-41).

The peptides are synthesized by a suitable method, such as byexclusively solid-phase techniques, by partial solid-phase techniques,by fragment condensation or by classical solution addition.

Common to chemical syntheses of peptides is the protection of the labileside chain groups of the various amino acid moieties with suitableprotecting groups which will prevent a chemical reaction from occurringat that site until the group is ultimately removed. Usually also commonis the protection of an alpha-amino group on an amino acid or a fragmentwhile that entity reacts at the carboxyl group, followed by theselective removal of the alpha-amino protecting group to allowsubsequent reaction to take place at that location. Accordingly, it iscommon that, as a step in the synthesis, an intermediate compound isproduced which includes each of the amino acid residues located in itsdesired sequence in the peptide chain with various of these residueshaving side-chain protecting groups.

The peptides are preferably prepared using solid phase synthesis, suchas that described by Burgess, K., Solid-Phase Organic Synthesis (JohnWiley & Sons 2000) and/or Merrifield, J. Am. Chem. Soc., 85, p 2149(1964). Thus, CRF analogs can be prepared in a straightforward mannerand then simply tested for biological activity, which facilitates theready preparation and evaluation of CRFR1 ligands. Solid-phase synthesisis commenced from the C-terminus of the peptide by coupling a protectedalpha-amino acid to a suitable resin as generally set forth in U.S. Pat.No. 4,244,946 issued Jan. 21, 1981 to Rivier et al. Starting materialfor a CRFR1 ligand can be prepared, e.g. by attaching α-amino-protectedIle to an MBHA resin.

After the desired amino acid sequence has been completed, theintermediate peptide is removed from the resin support unless it isdesired to form the cyclizing bond while attached to the resin, asdescribed hereinafter. Removal is effected by treatment with a reagent,such as liquid hydrogen fluoride (HF), which not only cleaves thepeptide from the resin but also cleaves all remaining side chainprotecting groups and the alpha-amino protecting group, if stillpresent, to obtain the peptide. When using hydrogen fluoride forcleaving, anisole or cresol and methylethyl sulfide are included in thereaction vessel as scavengers. When Met is present in the sequence, theBOC protecting group may be cleaved with trifluoroaceticacid(TFA)/ethanedithiol prior to cleaving the peptide from the resin toeliminate S-alkylation.

The cyclizing step for the CRF peptide analog depends, of course, uponthe type of linkage which is desired between the residues in the 31- and34-positions. To effect an amide cyclizing linkage (lactam bridge),cyclization may be carried out while the partially protected peptideremains attached to the resin as disclosed in U.S. Pat. Nos. 5,064,939and 5,043,322. Such a procedure effectively creates an amide cyclizingbond between the two desired side chains while other residues, such asAsp, Glu and/or Lys, in the peptide intermediate retain their side-chainprotection.

When cyclizing via an amide bond between a side-chain carboxyl group ofthe 31-position residue and a side-chain amino group of the 34-positionresidue, or vice-versa which is considered to be an equivalent linkage,it is preferable to synthesize the protected peptide on an MBHA or BHAresin and to derivatize the benzyl ester of the particular carboxyl acidside chain to the hydrazide while the peptide is still attached to theresin and then react it with a selectively deprotected amino-side chainas set forth in U.S. Pat. No. 5,043,322. Preferably cyclization isaccomplished by using a base-labile protecting group, e.g., OFm, for thecarboxyl side-chain of the residue to be involved in the amide-bondbridge and using Fmoc as a protecting group for the amino side chain onthe other residue that is to be involved. The α-amino protecting groupon the N-terminal residue, whether or not it is to be acylated, and allof the other side-chain protecting groups remain in place while the twobase-labile groups are removed using piperidine or the like. Followingthis selective removal, the reaction to accomplish cyclization iscarried out by treating with BOP which effects substantially completegeneration of the amide bond. Following cyclization, the peptide iscompletely deprotected and cleaved from the resin using a reagent, suchas HF. Optionally, a BOC-protecting group can be first removed from theN-terminus using TFA.

Alternatively, cyclizations of peptides by such amide linkages can alsobe effected using teachings of U.S. Pat. No. 4,115,554, (Sep. 19, 1978);U.S. Pat. No. 4,133,805 (Jan. 9, 1979); U.S. Pat. No. 4,140,767 (Feb.20, 1979); U.S. Pat. No. 4,161,521 (Jul. 17, 1979); U.S. Pat. No.4,191,754 (Mar. 4, 1980); U.S. Pat. No. 4,238,481 (Dec. 9, 1980); U.S.Pat. No. 4,244,947 (Jan. 13, 1981); and U.S. Pat. No. 4,261,885 (Apr.14, 1981).

Set forth hereinafter in the Examples are certain preferred methods forsynthesizing these peptides; however, those of skill in the art willreadily recognize techniques for synthesizing the invention peptides,see, e.g., Fmoc Solid Phase Peptide Synthesis: A Practical Approach(Oxford Univ Press, 2000); Marshak & Liu, Therapeutic Peptides andProteins : Formulation, Delivery, and Targeting (Current Communicationsin Molecular Biology) (Cold Spring Harbor Laboratory 1989); Cabilly, S.,Combinatorial Peptide Library Protocols, 1st edition (HumanaPress,1998); Crabb, J. W., Techniques in Protein Chemistry V (AcademicPress 1994), Lloyd-Williams et al., Chemical Approaches to the Synthesisof Peptides and Proteins (New Directions in Organic and BiologicalChemistry) (CRC Press 1997).

A straightforward assay can be carried out using rat anterior pituitarycells in monolayer culture to determine what CRF-activity a candidatepeptide will exhibit; the procedure which is used is that generally setforth in Endocrinology, 91, 562 (1972). The assay will show whether acandidate peptide will exhibit some activity as a CRF agonist andstimulate ACTH secretion by activating CRF receptors on such cells; inthis manner its intrinsic CRF activity is measured via the use of highdoses. A candidate CRFR1 ligand is also easily evaluated in a bindingassay using known CRF receptors, such as that described in Perrin, M.,et al., Endocrinology, 118, 1171-1179 (1986). CRF receptors and thedetails of binding assays are discussed later in this specification.Very generally, a binding assay may be carried out with human CRF-R1using a radioligand such as (cyclo 30-33)[I¹²⁵-D-Tyr¹², Glu³⁰, Lys³³,Nle^(21,38)]-r/hCRF(12-41) or its analog having D-His³², which have highaffinity for the human CRF-R1. For example, the first-named compound hasa K_(D) of 2.0 nanomolar (1.4-2.9) for binding to hCRFR1, which isessentially equal to that of the comparable D-Phe¹² analog. One suchrepresentative binding assay utilizing CRF-R1 receptor is described inChen, et al., P.N.A.S., 90, 8967-8971 (October 1993). Such assays areadvantageously used to screen for potential CRF-like ligands, in peptideor other form, using a labelled cyclic CRF analog, preferably such alabelled cyclic CRF agonist or antagonist with high affinity.

CRF receptors have now been cloned and are disclosed in theaforementioned Chen et al. article, in Perrin, M., et al., P.N.A.S, 92,2969-2973 (March 1995), and in Lovenberg, T., et al., P.N.A.S., 92,836-840 (January 1995). Binding affinity is a term used to refer to thestrength of interaction between ligand and receptor. To demonstratebinding affinity for a CRF receptor, the peptides of the invention areeasily evaluated using a tracer ligand of known affinity, such as¹²⁵I-radiolabelled oCRF or [D-Tyr¹², Nle^(21,38)]-r/hCRF(12-41), inbinding assay experiments which are well known in this art. The resultsof such assays indicate the affinity at which each ligand binds to a CRFreceptor, expressed in terms of K_(i), an inhibitory binding affinityconstant relative to such a known standard. K_(i) (inhibitory bindingaffinity constant) is determined using a “standard” or “tracer”radioactive ligand and thus measures the displacement of the tracer fromthe receptor or binding protein; it is most properly expressed withreference to such tracer. However, so long as these assays are carefullyperformed under specific conditions with relatively low concentrationsof receptor or the like, the calculated K_(i) will be substantially thesame as its dissociation constant K_(D). Dissociation constant K_(D) isrepresentative of the concentration of ligand necessary to occupyone-half (50%) of the binding sites of a receptor or the like. It isparticularly efficient to test for K_(i) because only a single tracerneed be labelled, e.g. radioiodinated. A given ligand having a highbinding affinity for a CRF receptor will require the presence of verylittle ligand to bind at least 50% of the available binding sites sothat the K_(D) value for that ligand and receptor will be a smallnumber. On the other hand, a given ligand having a low binding affinityfor a particular CRF receptor will require the presence of a relativelyhigh level of the ligand to bind 50% of the sites, so that the K_(D)value for that ligand and receptor will be a large number.

With respect to a particular receptor protein, a CRF analog peptidehaving a K_(D) of about 10 nM or less means that a concentration of theligand (i.e., the CRF analog peptide) of no greater than about 10 nMwill be required to occupy at least 50% of the active binding sites ofthe receptor protein. Such values may be fairly determined from theresults obtained using a radioiodinated standard and no more thanapproximately 0.8 nM of the receptor (approximately 10-20 pmolreceptor/mg membrane protein). Preferred peptides provided by thisinvention have a binding affinity (K_(D)) such that a ligandconcentration of about 10 nanomolar or less is required in order tooccupy (or bind to) at least 50% of the receptor binding sites, andthese are considered to have high affinity. Some of these CRF analogpeptides have a binding affinity of about 2 nM or less. Generally, forpurposes of this application, a dissociation constant of about 5nanomolar or lower is considered to be an indication of strong affinity,and a K_(D) of about 2 nanomolar or less is an indication of very strongaffinity. As mentioned above, it is considered to be particularlyadvantageous that these CRF analog peptides have a substantially higheraffinity for CRFR1 so that they are thus selective in their biologicaleffect.

These binding assays employing CRF receptors are straightforward toperform and can be readily carried out with initially identified orsynthesized peptides to determine whether such peptides will likely beeffective CRFR1 selective ligands. Such binding assays can be carriedout in a variety of ways as well known to one of skill in the art. Adetailed example of such an assay is set forth in the Perrin, M., etal., Endocrinology article. Moreover, the peptides of the presentinvention which incorporate a radioiodinated tyrosine residue areeffective tracers, selective to CRFR1, that may be used in highthroughput screenings.

The following Example 1 sets forth a preferred method for synthesizingCRFR1 ligands of interest by the solid-phase technique. These examplesare offered by way of illustration and not limitation.

EXAMPLE 1

The synthesis of (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), Glu³¹,Lys³⁴]-r/hCRF(4-41) having the amino acid sequence:Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH₂ is conducted in a stepwise manner on about 3 gramsof a MBHA hydrochloride resin, such as available from Bachem, Inc.,having a substitution range of about 0.1 to 0.5 mmoles/gm. resin. Thesynthesis is performed manually on an MBHA resin that has a substitutionof about 0.28 mequiv per gram of resin using a protocol such as thatwhich follows:

MIX TIMES STEP REAGENTS AND OPERATIONS MIN. 1 Methanol(MeOH) wash 1 210% TEA/DCM (v/v)wash 1 3 Methanol(MeOH) wash 1 4 DCM wash (3 times) 3 580 percent TFA plus 5 percent 10  m-cresol in CH₂Cl₂ 6 Methanol(MeOH)wash 1 7 TEA 10% in DCM 1 8 MeOH wash 1 9 TEA 10% in DCM 1 10 DCM wash(3 times) 3 11 BOC-amino acid (4 equiv. in 30 ml. of either DCM 20-30 orNMP depending upon the solubility of the particular protected aminoacid, (1 time) plus DIC (4 equiv) in CH₂Cl₂

After deprotection and neutralization, the peptide chain is builtstep-by-step on the resin. Generally, one to two mmol. of BOC-protectedamino acid in methylene chloride (DCM) is used per gram of resin (e.g. a2-5 fold excess depending on substitution of the resin), plus oneequivalent of 2 molar DIC in methylene chloride, for 20-30 minutes. WhenBOC-Arg(Tos) is being coupled, a mixture of 50% NMP and methylenechloride is used. Bzl is used as the hydroxyl side-chain protectinggroup for Ser and Thr. P-nitrophenyl ester(ONp) can be used to activatethe carboxyl end of Asn or Gln; for example, BOC-Asn(ONp) can be coupledovernight using one equivalent of HOBt in a 50% mixture of DMF andmethylene chloride. The amido group of Asn or Gln is protected by Xanwhen DIC coupling is used instead of the active ester method. 2-Cl-Z isused as the protecting group for the Lys side chain except for the Lysresidue which is to take part in the lactam bridge where Fmoc is used toprotect Lys³⁴. Tos is used to protect the guanidino group of Arg and theimidazole group of 5 His, and the side-chain carboxyl group of Glu orAsp is protected by OChx except for Glu³¹ which is protected by OFm. Atthe end of the synthesis, the following composition is obtained:

BOC-Pro-Pro-Ile-Ser(Bzl)-Leu-Asp(OChx)-Leu-Thr(Bzl)-D-Phe-His(Tos)-Leu-Leu-Arg(Tos)-Glu(OChx)-Val-Leu-Glu(OChx)-Nle-Ala-Arg(Tos)-Ala-Glu(OChx)-Gln(Xan)-Leu-Ala-Gln(Xan)-Gln(Xan)-Glu(OFm)-His(Tos)-Ser(Bzl)-Lys(Fmoc)-Arg(Tos)-Lys(2Cl-Z)-Leu-Nle-Glu(OChx)-Ile-Ile-resinsupport. Xan may have been partially or totally removed by TFA treatmentused to deblock the alpha-amino protecting group. The peptide-resin isthen treated with TFA to remove the BOC protecting group at theN-terminus. It is then reacted with acetic anhydride to acetylate theproline residue.

Cyclization (lactamization) of residues 31 and 34 is then performed bythe method referred to hereinbefore and described more fully as follows.After washes with dichloromethane(DCM) (2×) and 1-methyl-2-pyrrolidinone(NMP) (2×), the OFm/Fmoc groups of Glu³¹ and Lys³⁴, respectively, areremoved by 20% piperidine in NMP (1×1 min. and 2×10 min.), followed bywashing with NMP (2×), 10% ET₃N in DCM (v/v) (1×), methanol (MeOH) (2×)and DCM (2×). The peptide-resin is cyclized using a suitable couplingagent, e.g. by reaction at room temperature with twofold excess of HBTUor O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium borate (TBTU) inpresence of excess diisoproplyethylamine (DIEA) in NMP for 30 minutes.Other suitable reagents are well known and may also be used. Afterwashing, the cyclization may be repeated if desired to assurecompletion. The completion of the reaction is confirmed by the wellknown Kaiser ninhydrin test.

The resulting cyclic peptide-resin is cleaved and deprotected bytreatment with 1.0 g of p-cresol and 15 ml. hydrogen fluoride (HF) pergram of peptide-resin, first at −20° C. for 20 min. and then at 0° C.for one-half hour. After elimination of the HF under high vacuum, theresin and peptide are washed with dry diethyl ether, and the peptide isthen extracted with MeCN:H₂O(60:40) plus 0.1% TFA and separated from theresin by filtration.

The peptide is purified by preparative HPLC as described in Marki, etal., J. Am. Chem. Soc., 103, 3178 (1981); Rivier, et al., J.Chromatography, 288, 303-328 (1984); and Hoeger, et al.,BioChromatography, 2, 3, 134-142 (1987). The chromatographic fractionsare carefully monitored by HPLC, and only the fractions showingsubstantial purity are pooled.

To check whether the precise composition is achieved, the r/hCRF analogcan be hydrolyzed in sealed evacuated tubes containing constant boilingHCl, 3 μl of thioglycol/ml. and 1 nmol of Nle (as an internal standard)for 9 hours at 140° C. Amino acid analysis of the hydrolysates using aBeckman 121 MB amino acid analyzer shows amino acid ratios which confirmthat the 38-residue peptide structure has been obtained. MS analysis isemployed as reported hereinafter.

The peptide is judged to be homogeneous using reversed-phase highperformance liquid chromatography (RP-HPLC). It is specificallysubjected to RP-HPLC using a Waters HPLC system with a 0.46×25 cm.column packed with 5 μm Cl₁₈ silica, 300 Å pore size and TEAP buffers atdifferent pHs. Desalting of the purified peptide is achieved usingBuffer A which is an aqueous 0.1% trifluoroacetic acid solutionconsisting of 1.0 ml. of TFA per 1000 ml. of solution and Buffer B whichis 60% acetonitrile. It has a purity of about 98% measured by capillaryzone electrophoresis (CZE). Liquid secondary ion mass spectrometry(LSIMS) mass spectra are measured with a JEOL model JMS-HX110double-focusing mass spectrometer fitted with a Cs⁺ gun. An acceleratingvoltage of 10 kV and Cs⁺ gun voltage between 25 and 30 kV are employed.The measured value of 4471.33 obtained using LSIMS is in agreement withthe calculated value of 4470.53.

The synthesis is repeated omitting the cyclization step (i.e. byprotecting all Glu residues with OChx and all Lys residues with 2Cl-Z orsimply by deblocking the FMOC group with piperidine prior to HFtreatment) to produce a comparable linear peptide.

Binding assays with cells expressing human CRFR1 are carried out asdescribed in the Chen et al. P.N.A.S., supra. The affinities of testpeptides for CRFR1 and CRFR2 stably expressed in CHO cells weredetermined by competitive displacement of ¹²⁵I-(Nle²¹, Tyr³²) ovine CRF(for CRFR1) or of [¹²⁵I-Tyr^(o)-]Ucn (for CRFR2) as described. Data fromat least 3 experiments were pooled and inhibitory dissociation constant(K_(i)) values (95% confidence limits) were calculated using the LIGANDprogram of Munson and Rodbard (1980), Anal. Biochem, 107:220-239. Thecloned hCRFR1 binds the cyclic peptide with high affinity as determinedby the competitive displacement of bound radioligand. The K_(i) wasdetermined to be about 1.5 (0.9-2.6)nM, which may be compared to r/hCRFof about 0.95(0.47-2.0)nM. The linear peptide exhibits a K_(i) of 2.7(2.2-3.4)nmol. The difference is dramatic for similar stably transfectedCHO cells expressing human CRFR2 where the respective results for thecyclic and linear peptides were 224(140-370)nM and 500(330-770)nM.

The CRF agonists are examined for their effects on the secretion of ACTHand β-endorphin in vitro and also in vivo. In vitro potency to stimulatethe secretion of ACTH and β-endorphin by cultured rat pituitary cells ismeasured using the procedure generally set forth in Endocrinology, 91,562 (1972) and compared either against synthetic oCRF (the laboratorystandard) or against r/hCRF (an alternative standard). In vivo testingis carried out using the general procedure set forth in C. Rivier etal., Science, 218, 377 (1982). In vitro testing of the cyclic peptideshows a potency substantially greater that of the standard (oCRF),whereas the linear peptide shows a lesser potency but still greater thanthe standard. The cyclic peptide shows a significant lowering of bloodpressure when administered peripherally.

EXAMPLE 2

The peptide (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), Glu³¹,Lys³⁴]-oCRF(4-41) having the amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-Leu-Nle-Asp-Ile-Ala-NH₂ is synthesized using a procedure generally asset forth in Example 1. A portion of the peptide-resin is removed priorto cyclization, and it is cleaved and deprotected to provide thecorresponding linear peptide. The cyclic peptide strongly stimulates thesecretion of ACTH and β-END-LI and causes a very significant lowering ofblood pressure when administered peripherally. The linear peptide hasvery significantly lesser bioactivity. However, both peptides bindstrongly to CHO cells expressing CRFR1 and poorly to CHO cellsexpressing CRFR2.

EXAMPLE 3 A

The peptide (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(18,21), Glu³¹,Lys³⁴]-AHC(4-41) having the amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Glu-Ala-Leu-Lys-Arg-Leu-Leu-Leu-Glu-Glu-Ala-NH₂ is synthesized using a procedure generally asset forth in Example 1. A portion of the peptide-resin is removed priorto cyclization, and it is cleaved and deprotected so as to provide thecorresponding linear peptide. The cyclic peptide strongly stimulates thesecretion of ACTH and β-END-LI and causes a very significant lowering ofblood pressure when administered peripherally. The linear peptide hasvery significantly lesser bioactivity. Both peptides bind strongly toCRFR1 and only very weakly to CRFR2.

EXAMPLE 3 B

The peptide (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(18,21), Glu³¹,Lys³⁴]-sucker urotensin(4-41) having the amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Asn-Nle-Ile-Glu-Nle-Ala-Arg-Ile-Glu-Asn-Glu-Arg-Glu-Gln-Glu-Gly-Leu-Lys-Arg-Lys-Tyr-Leu-Asp-Glu-Val-NH₂ is synthesized using a procedure generally asset forth in Example 1.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 3 C

The peptide (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), Glu³¹,Lys³⁴]-porcine CRF(4-41) having the amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-Leu-Nle-Glu-Asn-Phe-NH₂ is synthesized using a procedure generally asset forth in Example 1.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 3 D

The peptide (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,37,38), Glu³¹,Lys³⁴]-fish CRF(4-41) having the amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Glu-Glu-His-Ser-Lys-Arg-Lys-Nle-Nle-Glu-Ile-Phe-NH₂ is synthesized using a procedure generally asset forth in Example 1.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 3 E

The peptide (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(14,18,24), Glu³¹,Lys³⁴]-maggy urotensin(4-41) having the amino acid sequence:

Ac-Pro-Pro-Nle-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Nle-Leu-Arg-Asn-Nle-Ile-His-Arg-Ala-Lys-Nle-Glu-Gly-Glu-Arg-Glu-Gln-Glu-Leu-Ile-Lys-Arg-Asn-Leu-Leu-Asp-Glu-Val-NH₂ is synthesized using a procedure generally asset forth in Example 1.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 3 F

The peptide (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(18,21), Glu³¹,Lys³⁴]-carp urotensin(4-41) having the amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Asn-Nle-Ile-Glu-Nle-Ala-Arg-Asn-Glu-Asn-Gln-Arg-Glu-Gln-Glu-Gly-Leu-Lys-Arg-Lys-Tyr-Leu-Asp-Glu-Val-NH₂ is synthesized using a procedure generally asset forth in Example 1.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 3 G

The peptide (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(14,18,24), Glu³¹,Lys³⁴]-flounder urotensin(4-41) having the amino acid sequence:

Ac-Pro-Pro-Nle-Ser-Ile-Asp-Leu-Thr-D-Phe-His-Nle-Leu-Arg-Asn-Nle-Ile-His-Arg-Ala-Lys-Nle-Glu-Gly-Glu-Arg-Glu-Gln-Glu-Gln-Ile-Lys-Arg-Asn-Leu-Leu-Asp-Glu-Val-NH₂ is synthesized using a procedure generally asset forth in Example 1.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 4

A synthesis as in Example 1 is performed, substituting Aib(2-aminoisobutyric acid) for Ser in the 33-position, to produce thefollowing peptide: (cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), Glu³¹,Aib³³, Lys³⁴]-r/hCRF(4-41), having the amino acid sequence:

(cyclo31-34)Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Glu-His-Aib-Lys-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH₂. A portion of thepeptide-resin is removed prior to cyclization, and it is cleaved anddeprotected to provide the corresponding linear peptide. The cyclicpeptide strongly stimulates the secretion of ACTH and β-END-LI andcauses a very significant lowering of blood pressure when administeredperipherally. The linear peptide has very significantly lesserbioactivity. Both peptides bind strongly to CRFR1 and only very weakerto CRFR2.

EXAMPLE 5

A synthesis as in Example 1 is carried out substituting C^(α)MeLeu forLeu¹⁵ to produce the following peptide: (cyclo 31-34)[Ac-Pro⁴, D-Phe¹²,CML¹⁵, Nle^(21,38), Glu³¹, Lys³⁴]-r/hCRF(4-41), having the amino acidsequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-CML-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH₂.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 5 A

A synthesis as in Example 1 is performed substituting C⁶⁰ MeLeu forLeu¹⁴ to produce the following peptide: (cyclo 31-34)[Ac-Pro⁴, D-Phe¹²,CML¹⁴, Nle^(21,38), Glu³¹, Lys³⁴]-r/hCRF(4-41), having the amino acidsequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-CML-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH₂. It

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 5 B

A synthesis as in Example 1 is carried out substituting C⁶⁰ MeLeu forLeu¹⁹ to produce the following peptide: (cyclo 31-34)[Ac-Pro⁴, CML¹⁹,Nle^(21,38), Glu³¹, Lys³⁴]-r/hCRF(4-41), having the amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-CML-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH₂.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 5 C

A synthesis as in Example 1 is performed substituting C^(α)MeLeu forLeu²⁷ to produce the following peptide: (cyclo 31-34)[Ac-Pro⁴, D-Phe¹²,Nle^(21,38), CML²⁷, Glu³¹, Lys³⁴]-r/hCRF(4-41), having the amino acidsequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-CML-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH₂.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 5 D

A synthesis as in Example 1 is performed substituting C^(α)MeLeu forLeu³⁷ to produce the following peptide: (cyclo 31-34)[Ac-Pro⁴, D-Phe¹²,Nle^(21,38), Glu³¹, Lys³⁴, CML³⁷]-r/hCRF(4-41), having the amino acidsequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-CML-Nle-Glu-Ile-Ile-NH₂.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 5 E

A synthesis as in Example 1 is carried out substituting C^(α)MeLeu forGlu¹⁷ to produce the following peptide: (cyclo 31-34)[Ac-Pro⁴, D-Phe¹²,CML¹⁷, Nle^(21,38), Glu³¹, Lys³⁴]-r/hCRF(4-41), having the amino acidsequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-CML-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH₂.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 5 F

The synthesis as in Example 1 is performed substituting C^(α)MeLeu forLeu²⁷ and D-His for His³² to produce the following peptide: (cyclo31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), CML²⁷, Glu³¹, D-His³²,Lys³⁴]-r/hCRF(4-41), having the formula:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-CML-Ala-Gln-Gln-Glu-D-His-Ser-Lys-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH₂.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 6 A

The synthesis of Example 5 C is repeated, but this time alsosubstituting C^(α)MeLeu for Leu¹⁴, to produce the following peptide:(cyclo 31-34)[Ac-Pro⁴, D-Phe¹², CML^(14,27), Nle^(21,38), Glu³¹,Lys³⁴]-r/hCRF(4-41), having the formula:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-CML-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-CML-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH₂.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 6 B

The synthesis of Example 5 C is repeated again, but this time alsosubstituting C^(α)MeLeu for Val¹⁸, to produce the following peptide:(cyclo 31-34)[Ac-Pro⁴, D-Phe¹², CML^(18,27), Nle^(21,38), Glu³¹,Lys³⁴]-r/hCRF(4-41), having the formula:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-CML-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH₂.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 6 C

The synthesis of Example 5 C is repeated once more, also substitutingC^(α)MeLeu for Lys³⁶, to produce the following peptide: (cyclo31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), CML^(27,36), Glu³¹,Lys³⁴]-r/hCRF(4-41), having the amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-CML-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-CML-Leu-Nle-Glu-Ile-Ile-NH₂.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

The above synthesis is generally repeated, substituting D-His for His³²,to produce the following peptide: (cyclo 31-34)[Ac-Pro⁴, D-Phe¹²,Nle^(21,38), CML^(27,36), Glu³¹, D-His³², Lys³⁴]-r/hCRF(4-41), havingthe amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-CML-Ala-Gln-Gln-Glu-D-His-Ser-Lys-Arg-CML-Leu-Nle-Glu-Ile-Ile-NH₂.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 6 D

The synthesis of Example 5 C is repeated, substituting C^(α)MeLeu forLeu³⁷, to produce the following peptide: (cyclo 31-34)[Ac-Pro⁴, D-Phe¹²,Nle^(21,38), CML^(27,37), Glu³¹, Lys³⁴]-r/hCRF(4-41), having the aminoacid sequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-CML-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-CML-Nle-Glu-Ile-Ile-NH₂.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 6 E

The synthesis of Example 5 C is repeated again, but this time alsosubstituting C^(α)MeLeu for Ile⁴⁰, to produce the following peptide:(cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), CML^(27,40), Glu³¹,Lys³⁴]-r/hCRF(4-41), having the amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-CML-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-Leu-Nle-Glu-CML-Ile-NH₂, and also its linear counterpart.

Purity of about 95% for the peptides is confirmed by HPLC and bycapillary zone electrophoresis (CZE) and identity is confirmed by massspectroscopy (MS). The measured value of 4498.32 obtained using liquidsecondary ion mass spectrometry (LSIMS) for the cyclic peptide is inagreement with the calculated value of 4498.56. The linear peptide has ameasured value of 4516.45 which corresponds to the calculated value of4516.57.

Binding assays with cells expressing human CRFR1 are carried out asdescribed with respect to Example 1. Data from at least 3 experimentsare pooled and inhibitory dissociation constant (K_(i)) values (95%confidence limits) are calculated using the LIGAND program of Munson andRodbard (1980), Anal. Biochem, 107:220-239. The cloned hCRFR1 binds thecyclic peptide with high affinity as determined by the competitivedisplacement of bound radioligand. The linear peptide exhibits aslightly higher K_(i). The difference is significant for similar stablytransfected CHO cells expressing human CRFR2 where the respectiveresults for the cyclic and linear peptides show both bind only weakly.The cyclic peptide stimulates the secretion of ACTH and β-END-LI andcauses a very significant lowering of blood pressure when administeredperipherally.

EXAMPLE 6 F

The synthesis of Example 5 C is repeated again, but this time alsosubstituting C⁶⁰ MeLeu for Ile⁴¹, to produce the following peptide:(cyclo 31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), CML^(27,41), Glu³¹,Lys³⁴]-r/hCRF(4-41), having the amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-CML-Ala-Gln-Gln-Glu-His-Ser-Lys-Arg-Lys-Leu-Nle-Glu-Ile-CML-NH₂.

The cyclic peptide binds strongly to CRFR1 and only very weakly toCRFR2. It stimulates the secretion of ACTH and β-END-LI and causes avery significant lowering of blood pressure when administeredperipherally.

EXAMPLE 6 G

The synthesis of Example 6 E is repeated, but this time alsosubstituting Aib for Ser³³, to produce the following peptide: (cyclo31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), CML^(27,40), Glu³¹, Aib³³,Lys³⁴]-r/hCRF(4-41), having the amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-CML-Ala-Gln-Gln-Glu-His-Aib-Lys-Arg-Lys-Leu-Nle-Glu-CML-Ile-NH₂, and also its linear counterpart.

Purity of about 98% for the peptides is confirmed by HPLC and bycapillary zone electrophoresis (CZE) and identity is confirmed by massspectroscopy (MS). The measured value of 4496.20 obtained using liquidsecondary ion mass spectrometry (LSIMS) for the cyclic peptide is inagreement with the calculated value of 4496.58. The linear peptide has ameasured value of 4514.45 which corresponds to the calculated value of4514.59.

Binding assays with cells expressing human CRFR1 are carried out asdescribed with respect to Example 1. The cloned hCRFR1 binds the cyclicpeptide with high affinity as determined by the competitive displacementof bound radioligand. The linear peptide exhibits a slightly higherK_(i). The difference is significant for similar stably transfected CHOcells expressing human CRFR2 where the respective results for the cyclicand linear peptides show both bind only weakly. The cyclic peptidestimulates the secretion of ACTH and β-END-LI and causes a verysignificant lowering of blood pressure when administered peripherally

EXAMPLE 6 H

The synthesis of Example 6 E is repeated, but this time alsosubstituting D-Ser for Ser³³, to produce the following peptide: (cyclo31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), CML^(27,40), Glu³¹, D-Ser³³,Lys³⁴]-r/hCRF(4-41), having the amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-CML-Ala-Gln-Gln-Glu-His-D-Ser-Lys-Arg-Lys-Leu-Nle-Glu-CML-Ile-NH₂, and also its linear counterpart.

Purity of about 96% for the peptides is confirmed by HPLC and bycapillary zone electrophoresis (CZE) and identity is confirmed by massspectroscopy (MS). The measured value of 4498.46 obtained using liquidsecondary ion mass spectrometry (LSIMS) for the cyclic peptide is inagreement with the calculated value of 4498.56. The linear peptide has ameasured value of 4516.27 which corresponds to the calculated value of4516.57.

Binding assays with cells expressing human CRFR1 are carried out asdescribed with respect to Example 1. The linear peptide exhibits aslightly higher K_(i). The difference is significant for similar stablytransfected CHO cells expressing human CRFR2 where the respectiveresults for the cyclic and linear peptides show both bind only weakly.The cyclic peptide stimulates the secretion of ACTH and β-END-LI andcauses a very significant lowering of blood pressure when administeredperipherally.

EXAMPLE 6 I

The synthesis of Example 6 E is repeated, but this time alsosubstituting D-Ala for Ser³³, to produce the following peptide: (cyclo31-34)[Ac-Pro⁴, D-Phe¹², Nle^(21,38), CML^(27,40), Glu³¹, D-Ala³³,Lys³⁴]-r/hCRF(4-41), having the amino acid sequence:

Ac-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-CML-Ala-Gln-Gln-Glu-His-D-Ala-Lys-Arg-Lys-Leu-Nle-Glu-CML-Ile-NH₂, and also its linear counterpart.

Purity of about 97% for the peptides is confirmed by HPLC and bycapillary zone electrophoresis (CZE) and identity is confirmed by massspectroscopy (MS). The measured value of 4482.47 obtained using liquidsecondary ion mass spectrometry (LSIMS) for the cyclic peptide is inagreement with the calculated value of 4482.57. The linear peptide has ameasured value of 4500.61 which corresponds to the calculated value of4500.58.

Binding assays with cells expressing human CRFR1 are carried out asdescribed with respect to Example 1. The cloned hCRFR1 binds the cyclicpeptide with high affinity as determined by the competitive displacementof bound radioligand. The linear peptide exhibits a slightly higherK_(i). The difference is significant for similar stably transfected CHOcells expressing human CRFR2 where the respective results for the cyclicand linear peptides show both bind only weakly. The cyclic peptidestimulates the secretion of ACTH and β-END-LI and causes a verysignificant lowering of blood pressure when administered peripherally.

CRF profoundly stimulates the pituitary-adrenalcortical axis, and actswithin the brain to mediate a wide range of stress responses. These CRFagonists should be useful to stimulate the functions of this axis insome types of patients with low endogenous glucocorticoid production;for example, they should be useful in restoring pituitary-adrenalfunction in patients having received exogenous glucocorticoid therapywhose pituitary-adrenalcortical functions remain suppressed.

Improving Learning and Memory

As noted above, the present invention provides methods for improvinglearning and memory through the administration to a patient of atherapeutically effective amount of a CRFR1 ligand. Such patients may beidentified through a clinical diagnosis based on symptoms of dementia orlearning and memory loss. Individuals with an amnesic disorder areimpaired in their ability to learn new information or are unable torecall previously learned information or past events. The memory deficitis most apparent on tasks to require spontaneous recall and may also beevident when the examiner provides stimuli for the person to recall at alater time. The memory disturbance must be sufficiently severe to causemarked impairment in social or occupational functioning and mustrepresent a significant decline from a previous level of functioning.The memory deficit may be age-related or the result of disease or othercause.

Dementia is characterized by multiple clinically significant deficits incognition that represent a significant change from a previous level offunctioning. Memory impairment involving inability to learn new materialor forgetting of previously learned material is required to make thediagnosis of a dementia. Memory can be formally tested by asking theperson to register, retain, recall and recognize information. Thediagnosis of dementia also requires at least one of the followingcognitive disturbances: aphasia, apraxia, agnosia or a disturbance inexecutive functioning. These deficits in language, motor performance,object recognition and abstract thinking, respectively, must besufficiently severe in conjunction with the memory deficit to causeimpairment in occupational or social functioning and must represent adecline from a previously higher level of functioning.

In addition, a number of biochemical tests that correlate levels of CRFwith impaired learning and memory may be utilized. For instance, thelevel of free CRF in the cerebrospinal fluid may be measured by ELISA orRIA. Additionally, or in place of the assays, brain imaging as describedwith a labeled ligand specific to the CRF-BP or CRF receptor may be usedto quantitate free receptor or CRF-BP, thus allowing one to know thatfree CRF is decreased. Finally, imaging of the brain with a ligandspecific to unbound CRF may be used to directly assay the amount of freeCRF in the brain.

The patient's minimental status is recorded by the Minimental Test forLearning and Memory, a standard test used by clinicians to determine ifa patient has impaired learning and memory (Folstein et al., J.Psychiatric Res. 12:185, 1975). This test involves a number of simpletasks and written questions. For instance, “paired-associate” learningability is impaired in amnesiac patients of several types includingthose suffering from head trauma, Korsakoffs disease or stroke (Squire,1987). Ten pairs of unrelated words (e.g., army-table) are read to thesubject. Subjects are then asked to recall the second word when giventhe first word of each pair. The measure of memory impairment is areduced number of paired-associate words recalled relative to a matchedcontrol group. This serves as an index of short-term, working memory ofthe kind that deteriorates rapidly in the early stages of dementing oramnesiac disorders.

Improvement in learning and memory constitutes either (a) astatistically significant difference between the performance ofligand-inhibitor treated patients as compared to members of a placebogroup; or (b) a statistically significant change in performance in thedirection of normality on measures pertinent to the disease model. Thisstrategy has been successfully employed in identifying therapeuticallyuseful cholinomimetics for memory improvement. Animal models or clinicalinstances of disease exhibit symptoms which are by definitiondistinguishable from normal controls. Thus, the measure of effectivepharmacotherapy will be a significant, but not necessarily complete,reversal of symptoms. Improvement can be facilitated in both animal andhuman models of memory pathology by clinically effective “cognitiveenhancing” drugs which serve to improve performance of a memory task.For example, cognitive enhancers which function as cholinomimeticreplacement therapies in patients suffering from dementia and memoryloss of the Alzheimer's type significantly improve short-term workingmemory in such paradigms as the paired-associate task (Davidson andStem, 1991). Another potential application for therapeutic interventionsagainst memory impairment is suggested by age-related deficits inperformance which are effectively modeled by the longitudinal study ofrecent memory in aging mice (Forster and Lal, 1992).

In animals, several established models of learning and memory areavailable to examine the beneficial cognitive enhancing effects andpotential anxiety-related side effects of activation of CRF-sensitiveneurons. The cognitive enhancing effects are measured by the Morris maze(Stewart and Morris, in Behavioral Ncuroscience, R. Saghal, Ed. (IRLPress, 1993) p. 107) the Y-maze (Brits et al., Brain Res. Bull. 6, 71(1981)), one-way active avoidance test, and two-way passive avoidancetest; anxiety-related effects are evaluated in the elevated plus-maze.(Pellow et al., J. Neurosci. Meth. 14:149,1985.)

The Morris water maze is one of the best validated models of learningand memory, and it is sensitive to the cognitive enhancing effects of avariety of pharmacological agents (McNamara and Skelton, Brain Res. Rev.18:33, 1993). The task performed in the maze is particularly sensitiveto manipulations of the hippocampus in the brain, an area of the brainimportant for spatial learning in animals and memory consolidation inhumans. Moreover, improvement in Morris water maze performance ispredictive of clinical efficacy of a compound as a cognitive enhancer.For example, treatment with cholinesterase inhibitors or selectivemuscarinic cholinergic agonists reverse learning deficits in the Morrismaze animal model of learning and memory, as well as in clinicalpopulations with dementia (McNamara and Skelton, 1993; Davidson andStem, 1991; McEntee and Crook, 1992; Dawson et al., 1992). In addition,this animal paradigm accurately models the increasing degree ofimpairment with advancing age (Levy et al., 1994) and the increasedvulnerability of the memory trace to pre-test delay or interference(Stewart and Morris, 1993) which is characteristic of amnesiac patients.

The test is a simple spatial learning task in which the animal is placedin tepid water, which is opaque due to the addition of powdered milk.The animals learn the location of the platform relative to visual cueslocated within the maze and the testing room; this learning is referredto as place learning.

As discussed in more detail below, 15 minutes prior to training on eachof days 1-3, groups of animals orally receive control solution or adosage of the ligand inhibitor. Control animals typically reach theplatform within five to ten seconds after three days of training. Themeasure of the memory modulator effects of a ligand inhibitor is a shiftof this time period. Administration of a ligand inhibitor results in adose-dependent increase in availability of synaptic CRF and a behavioraldose-dependent increase in acquisition and memory retention.

The Y-maze test based on visual discrimination is another assay oflearning and memory in animals. In this maze, two arms of the maze endin a translucent plastic panel behind which there is a 40-watt electricbulb. The start box is separated from the third arm by amanually-activated guillotine door. In the first trial, all animals areallowed to explore the maze for 5 minutes, and food pellets areavailable in each arm. On the second day, each animal is placed in thestart box with the door closed. When the door is opened, the animal isallowed to move down the arms and cat the pellets which are located inboth arms. On the third day, animals receive six trials in groups ofthree where one arm is closed at the choice point, no discriminativestimulus is present, and two food pellets are available in the open goalbox. On days 4-10, a light at the end of the arm with the food pelletsis illuminated and ten trials are run, again in groups of three. Thetime it takes for the animal to reach the food pellets is recorded.

The effectiveness of a ligand inhibitor to improve learning and memoryin the Y-maze is tested as follows. Fifteen minutes prior to each of theblocks of training trials on days 4-10, groups of animals orally receivecontrol solutions or doses of a ligand inhibitor. Control animals areexpected to make 50% correct choices. The measure of efficacy oftreatment on memory is an increase in correct responses.

The one-way active avoidance test is another assay of learning andmemory in animals. It may be used to assess improvement in age-relatedmemory deficits. An animal is placed in a footshock compartment; anopening door to a safe compartment serves as a signal for avoidance.Briefly, in this test an animal is placed in a Skinner box enclosurethat contains a grid floor composed of stainless steel bars. A sevenwatt light and tone generator at each end of the box serve asconditioned stimuli. A rat or mouse is initially trained by being placedin the footshock compartment facing away from the door. A shock isadministered simultaneously with the door opening to the safecompartment. At intervals, the test is repeated, only the shock isdelayed for 10 seconds after the door is opened. The time it takes theanimal to leave the footshock compartment is recorded.

The effectiveness of a ligand inhibitor to improve memory and learningin the one-way avoidance or control solution is tested as follows.Animals are given the ligand inhibitor 15 minutes prior to training.Twenty-four hrs later, the groups are tested for retention, withoutfurther administration of ligand inhibitor. The measure of efficacy is ashortened latency time to leaving the footshock compartment.

The two-way passive avoidance test is another assay of learning andmemory. An animal is placed in the safe compartment of the Skinner boxand when it enters the footshock compartment, the door is closed and amild shock is administered. The latency time for entering the secondcompartment is recorded. Memory is tested 1 to 7 days later. At thistime, a shock is not administered.

The effectiveness of a ligand inhibitor to improve learning and memoryis tested as follows. Immediately prior to training, groups of animalsorally receive control solutions or doses of ligand inhibitor. Latencytime for entering the footshock compartment is then determined.

The elevated plus maze test measures anxiogenic responses in anapproach-avoidance situation involving an exposed, lighted space versusa dark, enclosed space. Both spaces are elevated and are set up as tworunways intersecting in the form of a plus sign. This type ofapproach-avoidance situation is a classical test of “emotionality” andis very sensitive to treatments that produce disinhibition and stress.Animals are placed in the center of the maze and are allowed free accessto all four arms in a five minute testing period. The time spent in eacharm is recorded.

In humans, methods for improving learning and memory may be measured bysuch tests as the Wechsler Memory Scale or a pair-associate memory task.The Wechsler Memory Scale is a widely-used pencil-and-paper test ofcognitive function and memory capacity. In the normal population, thestandardized test yields a mean of 100 and a standard deviation of 15,so that a mild amnesia can be detected with a 10-15 point reduction inthe score, a more severe amnesia with a 20-30 point reduction, and soforth (Squire, 1987). During the clinical interview, a battery of tests,including, but not limited to, the Minimental test, the Wechsler memoryscale, or paired-associate learning are applied to diagnose symptomaticmemory loss. These tests provide general sensitivity to both generalcognitive impairment and specific loss of learning/memory capacity(Squire, 1987). Apart from the specific diagnosis of dementia oramnestic disorders, these clinical instruments also identify age-relatedcognitive decline which reflects an objective diminution in mentalfunction consequent to the aging process that is within normal limitsgiven the person's age (DSM IV, 1994). As noted above, “improvement” inlearning and memory is present within the context of the presentinvention if there is a statistically significant difference in thedirection of normality in the paired-associate test, for example,between the performance of ligand-inhibitor treated patients as comparedto members of the placebo group or between subsequent tests given to thesame patient.

Decreasing Food Intake

As noted above, the present invention provides methods for decreasingfood intake through the administration to a patient of a therapeuticallyeffective amount of a CRFR1 ligand. CRF has been shown to be animportant modulator of food intake. For example, administration of CRFagonists or conditions that elevate endogenous CRF levels (e.g., stress)diminish food intake (Appel et al., Endoc. 128:3237, 1991; Krahn andGosnell, Psychiat. Med. 7:235, 1989; McCarthy et al., Am. J. Physiol.264:E638, 1993). Thus, administration of CRF causes significant decreaseon nocturnal food intake (Gosnell et al., Peptides 4:807, 1983), loweredbody weight in rats (Hotta et al., Life Sci. 48:1483, 1991) andincreased temperature response in brown adipose tissue (LeFeuvre et al.,Neuropharmacol. 26:1217, 1987). Furthermore, neuropeptide Y (NPY), whichis the strongest known stimulus of food intake, can be potentiated inits effect upon co-administration of a ligand of the CRF receptor.

Patients may be identified by being obese. An obese individual weighsmore than a target weight considered normal for that person's age,gender and height and can be identified objectively by a body mass index(BMI-calculated as weight in kilograms/height in meters²) at or higherthan the 85th percentile of the same reference population (NationalCenter for Health Statistics, “Obese and Overweight Adults in the UnitedStates.” Series 11, No. B0, U.S. Government Printing Office, Washington,D.C., 1983). In addition, evidence that CRF is involved for a particularindividual may be obtained by demonstrating decreased CRF levels in thecerebrospinal fluid or by brain imaging as described above. Because thehypothalamus is a common brain area mediating the effects of CRF on foodintake and endocrine parameters, alterations in pituitary hormoneconcentration may also reflect altered levels in hypothalamic CRF.

A decrease in food intake may be measured both in the delayed initiationof a meal and the reduction in the overall duration or quantity of foodconsumption. Smith, “Satiety and the Problem of Motivation,” in D. W.Pfaff (ed.), The Physiological Mechanisms of Motivation,Springer-Verlag, New York, pp. 133-143, 1982. In addition, the selectionof particular nutrients in a food choice situation serves as asupplemental measure of specific hunger (Rozin, Adv. Study Behav. 6:21,1976).

There are two established animal models of appetite regulation. One is asimple measurement of food intake, and the second is a measurement ofdiet self-selection in a cafeteria environment. In the first method,food intake is limited for 24 hours followed by two hours of access to apreweighed portion of laboratory chow in the animal's home cage. Foodintake is measured at 60 and 120 minutes by weighing the remainingpellets. These tests may also be performed on animals that are obese dueto genetic mutations and which effectively reproduce symptoms ofovereating and deranged nutrient selection (Argiles, Prog. Lipid Res.28:53, 1989; Wilding et al., Endocrinol. 132:1939, 1993).

In the cafeteria environment, diets are specially formulated withdiffering proportions of macronutrients, such as carbohydrate, protein,and fat, so as to measure preference for specific nutrients based onsensory attractiveness or post-ingestive benefit. Diet selection isaltered, in part, by a wide variety of neurochemical systems. Thesetests are useful for detection of subtle changes in food intakeregulation which impact phenomena, such as craving or binging, and arerelevant for the diagnosis of eating disorders, such as anorexia nervosaand obesity. Following establishment of a baseline for animals, 15minutes prior to testing each animal receives an oral dose of a ligandinhibitor. Food intake is measured as described for the feeding test orthe diet self-selection in the cafeteria environment, and test resultsare compared to baseline. In addition, overeating in an animal model ofnicotine withdrawal and in genetically obese rats (Zucker strain)provide other models to test the effect of a ligand inhibitor onappetite regulation. Briefly, in the nicotine withdrawal model, animalsare administered nicotine in a chronic fashion. These animals showinhibition of normal weight gain and reduction of food and water intake.Upon cessation of nicotine treatment, animals significantly increaseboth body weight and intake of food and water. The effect of ligandinhibitors on appetite during nicotine withdrawal is assessed byadministering the ligand inhibitor three days following nicotinecessation.

A genetic basis for overeating has been discovered in both mice (e.g.,ob/ob) and rats (Zucker strain; fa/fa). These animals offer other modelsof overeating to assess the efficacy of ligand inhibitors. Inparticular, Zucker rats are used as subjects. Groups of rats are treatedwith vehicle or ligand inhibitor on a daily basis over a set timeperiod, such as one week. Subsequent weight gain or food intake ismeasured. Normal Zucker rats (not genetically obese) serve as controls.Administration of a ligand inhibitor reduces food intake and body weightgain relative to that of normal rats.

In humans, obesity is related not only to overeating, but may also berelated to consumption of nutritionally imbalanced diets such as adisproportionately large intake of sweet or fatty foods. (Drewnowski etal., Am. J. Clin. Nutr. 46:442, 1987.) Thus, clinical manifestations ofappetite regulation are readily detected using controlled experimentaldiets or cafeteria self-selection protocols which record intake patternsin terms of quantity, meal duration, and choice (Kissileff, Neurosci.Biobehav. Rev. 8:129, 1984). In these tests, following a baselinedetermination for each individual, measurement of food intake orself-selection in the cafeteria environment are measured. Improvement inthe context of the treatment of obesity constitutes a weight loss orreduction in food intake exhibited by treated patients as compared tomembers of a placebo group. Moreover, this strategy has been successfulin identifying serotonergic agonists for obesity.

Diseases Associated with Low Levels of CRF

As noted above, the present invention provides methods for treatingdiseases associated with low levels of CRF through the administration toa patient of a therapeutically effective amount of a ligand inhibitor ofa CRF/CRF-BP complex. Such patients may be identified through diagnosisof eating disorders, neuroendocrine disorders, and cognitive disorders,such as Alzheimer's disease. In addition, other conditions associatedwith decreased CRF levels, such as atypical depression, seasonaldepression, chronic fatigue syndrome, obesity, vulnerability toinflammation disease, post-traumatic stress disorder, andpsychostimulant withdrawal often present a profile of hypothyroidism anddecreased stress system activity which is identified characteristicallyby a decrease in urinary free cortisol and plasma ACTH. Thus, thesediseases and conditions would likely be resolved in part by restorationor potentiation of brain CRF levels (Chrousos and Gold, JAMA 267:1244,1992).

The hallmark of this diverse set of human disease states isdysregulation of the pituitary-adrenal axis with a presumed derangementof brain CRF. Hence, the fact that experimental alternation ofCRF/pituitary-adrenal systems in laboratory animals reproduces essentialfeatures of the above syndromes, namely behavioral despair (Pepin etal., 1992), exercise fatigue (Rivest and Richard, 1990), obesity(Rothwell, 1989) and hyperarousal associated with psychostimulantwithdrawal (Koob et al., 1993; Swerdlow et al., 1991) suggests the broadutility of pharmacotherapies designed to normalize endogenous levels ofCRF.

The essential feature of seasonal depression (major depressive disorderwith seasonal pattern) is the onset and remission of major depressiveepisodes at characteristic times of the year. In most cases, theepisodes begin in fall or winter and remit in spring. Major depressiveepisodes that occur in a seasonal pattern are often characterized byprominent anergy, hypersomnia, overeating, weight gain, and a cravingfor carbohydrates and must persist for a period of at least two weeksduring which there is either depressed mood or the loss of interest orpleasure in nearly all activities.

The essential feature of post-traumatic stress disorder is thedevelopment of characteristic symptoms following exposure to an extremetraumatic stressor involving direct personal experience of an event thatinvolves actual or threatened death or serious injury to one's own oranother's physical integrity. The person's response to the event mustinvolve intense fear, helplessness, or horror. The traumatic event isreexperienced as intrusive recollections or nightmares which triggerintense psychological distress or physiological reactivity. The fullsymptom picture must be present for more than one month and causeclinically significant distress or impairment in social or occupationalfunctioning.

The essential feature of nicotine withdrawal (nicotine-induced disorder)is the presence of a characteristic withdrawal syndrome that developsafter the abrupt cessation of, or reduction in, the use ofnicotine-containing products following a prolonged period (at leastseveral weeks) of daily use. Diagnosis of nicotine withdrawal requiresidentification of four or more of the following: dysphoric or depressedmood, insomnia, irritability or anger, anxiety, difficultyconcentrating, restlessness or impatience, decreased heart rate andincreased appetite or weight gain. These symptoms must cause clinicallysignificant distress or impairment in social, occupational functioning.

Improvement constitutes either (a) a statistically significant change inthe symptomatic condition of a treated individual as compared to abaseline or pretreatment condition on measures pertinent to the diseasemodel; or (b) a statistically significant difference in the symptomaticcondition of ligand-inhibitor treated patients and members of a placebogroup. Clinical instances of disease exhibit symptoms which are, bydefinition, distinguishable from normal controls. For depression,several rating scales of depression are used. (See Klerman et al.,Clinical Evaluation of Psychotropic Drugs: Principles and Guidelines,Prien and Robinson (eds.), Raven Press, Ltd., New York, 1994). One test,the Hamilton Rating Scale for Depression, is widely used to evaluatedepression and is also used to assess symptom changes in response totreatment. Other tests and ratings can be found in the DSM-IV manual.For nicotine withdrawal, as well as the other disorders, tests forevaluation of the severity of the disorder can be found in the DSM-IVmanual.

Alzheimer's Disease

As noted above, the present invention provides methods for treatingAlzheimer's disease (“AD”) through the administration to a patient of atherapeutically effective amount of a CRFR1 ligand. Such patients may beidentified through clinical diagnosis based on symptoms of dementia orlearning and memory loss which are not attributable to other causes. Inaddition, patients are also identified through diagnosis of brainatrophy as determined by magnetic resonance imaging.

Decreased levels of CRF are shown to be implicated in Alzheimer'sdisease. Brains obtained post-mortem from ten individuals with AD andten neurologically normal controls were chosen for study. Standard areasof frontal pole, parietal pole, temporal pole, and occipital pole weredissected from fresh brain, frozen in dry ice, and stored at −70° C.until they were processed for CRF radioimmunoassay and CRF-BP assay.Formalin-fixed samples of the cerebral cortex and hippocampus wereembedded in paraffin and subsequently sectioned and stained withhematoxylin/eosin and silver impregnation. Examination of stainedsections from brains of AD patients showed abundant neuritic plaques andneurofibrillary tangles typical of AD, whereas control cases showednone.

Several established animal models of Alzheimer's disease which focus oncholinergic deficits are available. The primary role of cholinergicdeficits in AD is well established. In AD, there are significantpositive correlations between reduced choline acetyltransferase activityand reduced CRF levels in the frontal, occipital, and temporal lobes(DeSouza et al., 1986). Similarly, there are negative correlationsbetween decreased choline acetyltransferase activity and an increasednumber of CRF receptors in these three cortices (Id.). In two otherneurodegenerative diseases, there are highly significant correlationsbetween CRF and choline acetyltransferase activity in Parkinson'sdisease, but only a slight correlation in progressive supranuclear palsy(Whitehouse et al., 1987).

In rats, anatomic and behavioral studies evidence interactions betweenCRF and cholinergic systems. First, in some brain stem nuclei, CRF andacetylcholinesterase are co-localized, and some cholinergic neurons alsocontain CRF. Second, CRF inhibits carbachol-induced behaviors (carbacholis a muscarinic cholinergic receptor antagonist), suggesting that CRFhas effects on cholinergic systems (Crawley et al., Peptides 6:891,1985). Treatment with another muscarinic cholinergic receptorantagonist, atropine, results in an increase in CRF receptors (DeSouzaand Battaglia, Brain Res. 397:401, 1986). Taken together, these datashow that CRF and cholinergic systems interact similarly in humans andanimals.

An animal model of Alzheimer's disease which focuses on cholinergicdeficits is produced by the administration of scopolamine, anon-selective postsynaptic muscarinic receptor antagonist that blocksthe stimulation of postsynaptic receptors by acetylcholine. In theseanimals, memory deficits are readily apparent as measured by passiveavoidance or delayed-matching-to-position tests, which distinguish motoror perceptual deficits from amnesia or cognitive enhancing effects ofexperimental treatments. Thus, the Morris maze and Y-maze testsfollowing scopolamine-induced amnesia are utilized to test memoryimpairment and subsequent enhancement following administration of ligandinhibitor. In the Morris maze, the design of the experiment isessentially as described above, but is modified to include treatment 30minutes prior to training on each of days 1 to 3 with an ip injection ofscopolamine hydrobromide (0.3 mg/kg). This amnestic dose of scopolamineimpairs acquisition and retention of spatial and avoidance learningparadigms in the rat. The anti-amnestic effects of a ligand inhibitorare measured relative to the concurrent control groups who receive or donot receive scopolamine. The effect of the ligand inhibitors on reversalof scopolamine-induced amnesia using the Y-maze is performed similarlyto the Y-maze test described above. Modification of this test includestreatment 30 minutes prior to training on days 5 to 10 with an ipinjection of scopolamine hydrobromide (0.3 mg/kg). The anti-amnesticeffects of a ligand inhibitor administered centrally or systemically aremeasured relative to concurrent control and scopolamine treated-controlgroups.

Several tests measuring cognitive behavior in AD have been designed.(See Gershon et al., Clinical Evaluation of Psychotropic Drugs:Principles and Guidelines, Prien and Robinson (eds.), Raven Press, Ltd.,New York, 1994, p. 467.) One of these tests, BCRS, measuresconcentration, recent memory, past memory, orientation, and functioningand self-care. The BCRS is designed to measure only cognitive functions.This test, as well as the Weschler Memory Scale and the Alzheimer'sDisease-Associated Scale, may be used to determine improvement followingtherapeutic treatment with ligand inhibition. As noted above,“improvement” in Alzheimer's disease is present within the context ofthe present invention if there is a statistically significant differencein the direction of normality in the Weschler Memory Scale test, forexample, between the performance of ligand-inhibitor treated patients ascompared to members of the placebo group or between subsequent testsgiven to the same patient. In addition, scopolamine-induced amnesia inhumans can be used as a model system to test the efficacy of the ligandinhibitors.

The CRFR1 ligand peptides of the invention will also be therapeuticallyuseful to modulate blood flow in many various vascular beds, andparticularly in desired tissues and organs. They should be of use forincreasing blood flow to the gastrointestinal tract of animals,particularly humans and other mammals, as they are expected to dilatethe mesenteric vascular bed. CRF has been shown to modulate vascularpermeability (Wei E. T. et al., “Peripheral anti-inflammatory actions ofcorticotropin-releasing factor”, pp. 258-276, Corticotropin-ReleasingFactor (Ciba Foundation Symposium 172) John Wiley & Sons, 1993), andthese CRFR1 ligands will also reduce vascular leakage and have asalutary effect on injury- or surgery-induced tissue swelling andinflammation. Therefore, these CRFR1 ligands can be administeredparenterally to decrease inflammation, swelling and edema and to reducefluid loss following heat injury.

oCRF, r/hCRF, urotensin I and sauvagine have been shown to inhibitgastric acid production, and the CRFR1 ligands of the invention areconsidered to also likely be effective in the treatment of gastriculcers by reducing gastric acid production and/or inhibiting certaingastrointestinal functions in a mammal.

These CRFR1 ligand peptides may also be used to evaluate hypothalamicpituitary adrenal function in mammals with suspected endocrine orcentral nervous system pathology by suitable administration followed bymonitoring bodily functions. For example, administration may be used asa diagnostic tool to evaluate Cushing's disease and affective disorders,such as depressive illness.

CRFR1 ligands or the nontoxic addition salts thereof would normally beadministered to mammals, including humans combined with apharmaceutically or veterinarily acceptable carrier. As used herein, theterms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably. Preferably, thematerials are capable of administration to a mammal without theproduction of undesirable physiological effects, such as nausea,dizziness, gastric upset and the like. The peptides should be at leastabout 90% pure and preferably should have a purity of at least about97%. Administration to a patient would be in a therapeutically effectiveamount, which is an amount calculated to achieve the desired effect,either increasing the level of free CRF in the brain, improving learningand memory, decreasing food intake, activating CRF neurocircuitry in thebrain, treating diseases associated with low levels of CRF in the brain,treating the symptoms associated with Alzheimer's disease, treatingobesity, treating atypical depression, treating substance abusewithdrawal, treating post-partum depression, or age-related memory loss.It will be apparent to one skilled in the art that the route ofadministration may vary with the particular treatment and also withwhether a peptide or non-peptide ligand inhibitor is administered.Routes of administration may be either non-invasive or invasive.Non-invasive routes of administration include oral, buccal/sublingual,rectal, nasal, topical (including transdermal and ophthalmic), vaginal,intravesical, and pulmonary. Invasive routes of administration includeICV, intraarterial, intravenous, intradermal, intramuscular,subcutaneous, intraperitoneal, intrathecal and intraocular.

Intracerebroventricular (ICV) injections are performed on animals asfollows. Animals are anesthetized with halothane and secured in a KOPFstereotaxic instrument. A guide cannula aimed above the lateralventricle is implanted and anchored to the skull with two stainlesssteel screws and dental cement. For injections, a 30 gauge stainlesssteel cannula attached to 60 cm of PE 10 tubing is inserted through theguide to 1 mm beyond its tip. Two microliters of ligand inhibitor areinjected by gravity flow over a one minute period simply by raising thetubing above the head of the animal until flow begins. Procedures forthe other routes of administration are well known in the art.

The required dosage will vary with the particular condition beingtreated, with the severity of the condition and with the duration ofdesired treatment, and multiple dosages may be used for a single day.For parental administration, solutions in peanut oil, in aqueouspropylene glycol, or in sterile aqueous solution may be employed. Suchaqueous solutions, which are suitably buffered, are especially suitablefor intravenous, intramuscular, subcutaneous (s.c.) and intraperitonealadministration. Sterile aqueous media are readily available, and fors.c. administration, corn oil or a 3-6% mannitol solution may bepreferred. Such peptides are often administered in the form ofpharmaceutically acceptable nontoxic salts, such as acid addition saltsor metal complexes. The salts of trifluoroacetic acid and pamoic acidmay be preferred.

The peptides should be administered under the guidance of a physician insingle or multiple doses, and pharmaceutical compositions will usuallycontain the peptide in conjunction with a conventional,pharmaceutically-acceptable carrier. These carriers are well known inthe art and typically contain non-toxic salts and buffers. Such carriersmay comprise buffers like physiologically-buffered saline,phosphate-buffered saline, carbohydrates such as glucose, mannose,sucrose, mannitol or dextrans, amino acids such as glycine,antioxidants, chelating agents such as EDTA or glutathione, adjuvantsand preservatives. Acceptable nontoxic salts include acid addition saltsor metal complexes, e.g., with zinc, iron, calcium, barium, magnesium,aluminum or the like (which are considered as addition salts forpurposes of this application). Illustrative of such acid addition saltsare hydrochloride, hydrobromide, sulphate, phosphate, tannate, oxalate,fumarate, gluconate, alginate, maleate, acetate, citrate, benzoate,succinate, malate, ascorbate, tartrate and the like. If the activeingredient is to be administered in tablet form, the tablet may containa binder, such as tragacanth, corn starch or gelatin; a disintegratingagent, such as alginic acid; and a lubricant, such as magnesiumstearate. If administration in liquid form is desired, sweetening and/orflavoring may be used, and intravenous administration in isotonicsaline, phosphate buffer solutions or the like may be effected.

The effective dosage generally depends on the intended route ofadministration and other factors such as age and weight of the patient,as generally known to a physician, and also upon the illness beingtreated. Usually, the dosage will be from about 0.01 to about 10milligrams of the peptide per kilogram of the body weight of the hostanimal per day. For the treatment of certain indications daily dosagesup to about 100 mg/kg may be employed. The daily dosage may be given ina single dose or up to three divided doses.

As mentioned hereinbefore, CRF receptors have now been cloned andbinding affinity tests and binding assays employing CRF receptors arereadily carried out with initially identified or synthesized peptides todetermine whether such peptides will likely be effective CRFR1 ligandsas described in WO 96/18649. Such receptor assays can be used as screensfor potential drugs which interact with CRF and/or CRF receptors.

As used herein all temperatures are ° C. and all ratios are by volume.Percentages of liquid materials are also by volume.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventor, it should be understood that various changes and modificationsas would be obvious to one having the ordinary skill in this art may bemade without departing from the scope of the invention which is setforth in the claims appended hereto. The disclosures of all previouslymentioned U.S. patents are expressly incorporated herein by reference.

1. A 38-residue or 39-residue CRF cyclic peptide, or a nontoxic saltthereof, which binds to CRFR1 with an affinity substantially greaterthan it binds to CRFR2, which peptide has the following formula:

wherein the sidechains of Glu and Lys indicated are covalently linked;Y₁ is an acyl group having not more than 15 carbon atoms or isradioiodinated tyrosine; R₁₄ is CML or Leu; R₁₅ is CML or Leu; R₁₇ isGlu or CML; R₁₈ is Val or CML; R₁₉ is CML or Leu; R₂₇ is CML or Leu; R₃₂is His or D-His; R₃₃ is Aib, D-Ala, D-Ser or Ser; R₃₆ is Lys or CML; R₃₇is CML or Leu; R₄₀ is Ile or CML; and R₄₁ is Ile or CML; provided thatD-β-(2-napthyl)alanine(D-2Nal) or D-Leu may be substituted for D-Phe. 2.A CRF agonist peptide, or a nontoxic salt thereof, which binds to CRFR1with an affinity substantially greater than it binds to CRFR2, whichpeptide has the following formula:

wherein Y₁ is an acyl group having not more than 7 carbon atoms or isradioiodinated tyrosine, and wherein a cyclizing bond may exist betweenthe side chains of Glu and Lys as indicated.
 3. A 38-residue or39-residue CRFR1 ligand cyclic peptide which binds to CRFR1 with anaffinity substantially greater than it binds to CRFR2, which peptide hasthe following formula, or a nontoxic salt thereof:

wherein the side chains of Glu and R₃₄ are covalently linked asindicated; Y₁ is an acyl group having not more than 7 carbon atoms or isradioiodinated tyrosine; R₆ is Ile, Met or Nle; R₈ is Leu or Ile; R₁₁ isThr or Ser; R₁₄ is CML or Leu; R₁₅ is Leu or CML; R₁₈ is Val, CML, Nleor Met; R₂₀ is Glu or D-Glu; R₂₂ is Ala or Thr; R₂₃ is Arg or Lys; R₂₅is Asp or Glu; R₂₇ is Leu or CML; R₂₉ is Gln or Glu; R₃₂ is His, Aib,Ala, Gly, Leu, Gln or Glu; R₃₃ is Aib or an L- or D-isomer of Ser, Asn,Leu, Ala, CML or Ile; R₃₄ is Lys or Orn; R₃₆ is Lys or Leu; R₃₇ is CMLor Leu; R₃₉ is Glu or Asp; R₄₀ is Ile, CML or Glu; and R₄₁ is Ala, Aibor Ile; provided that D-β-(2-napthyl)alanine(D-2Nal) or D-Leu may besubstituted for D-Phe.
 4. A CRF cyclic peptide according to claim 3having the formula:

wherein Y₁ is an acyl group having not more than 7 carbon atoms; R₂₀ isGlu or D-Glu; R₂₂ is Ala or Thr; R₂₃ is Arg or Lys; R₂₉ is Gln or Glu;R₃₂ is His, Aib or Ala; R₃₆ is Lys or Leu; R₃₇ is Leu or CML; R₃₉ is Gluor Asp; R₄₀ is Ile, CML or Glu; and R₄₁ is Ile, Aib or Ala; wherein theremaining variabies are as defined in claim
 3. 5. A peptide according toclaim 3 wherein R₁₈ is Val, R₂₂ is Ala, R₂₃ is Arg, R₂₅ is Glu, R₃₉ isGlu, and R₄₁ is Ile.
 6. A peptide according to claim 3 having thefollowing formula, or a nontoxic salt thereof:

wherein Y₁ is an acyl group having not more than 7 carbon atoms; R₂₂ isAla or Thr; R₂₃ is Arg or Lys; R₂₇ is Leu or CML; R₃₂ is His; R₃₃ is Seror Aib; and R₄₀ is Ile or CML.
 7. A peptide according to claim 1 havingthe formula:


8. A peptide according to claim 1 having the formula:


9. A peptide having the formula: